Recombinant Anti-SARS-CoV-2 nucleocapsid protein antibody [EPR24334-118] (ab271180)

Blocking and diluting buffer and concentration: 5% NFDM/TBST.

Exposure time 7.75 seconds

Blocking and diluting buffer and concentration: 5% NFDM/TBST.

Data was kindly provided by Dr. Hangping Yao, State Key Laboratory for Diagnosis and Treatment of Infectious Diseases in Zhejiang University, School of Medicine.

Samples

Lanes 1 & 3: Recombinant Human coronavirus SARS-CoV-2 Nucleocapsid Protein (His tag) (ab273530) (0.2 µg)

Lanes 2 & 4: Recombinant Human coronavirus SARS-CoV-2 Nucleocapsid Protein (His tag) (ab272107) (0.2 µg)

Green: Rabbit monoclonal [EPR24334-118] to SARS-CoV-2 nucleocapsid protein (ab271180)

Red: Mouse monoclonal [HIS.H8] to 6X His tag® (ab18184)

Lanes 1 & 2: Green - ab271180 observed at 50 kDa. 

Lanes 3 & 4: Red - loading control Mouse anti-6x HisTag observed at 50 kDa.

ab271180 was shown to react with SARS-CoV-2 nucleocapsid protein in Western blot. Membranes were blocked in 3% milk before incubation with ab271180 and Mouse anti-6x HisTag (ab18184) overnight at 4 °C at a 1/1000 dilution. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) secondary antibodies at 1/20000 dilution for 1 h at room temperature before imaging.

Immunohistochemical analysis of paraffin-embedded Human lung labeling SARS-CoV-2 with ab271180 at 1/4000 dilution followed by ready to use Goat Anti-Rabbit IgG H&L (HRP polymer). Negative control: No staining on human lung. The section was incubated with ab271180 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Goat Anti-Rabbit IgG H&L (HRP polymer).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

Immunohistochemical analysis of paraffin-embedded HEK-293T (human epithelial cell line from embryonic kidney transformed with large T antigen) transfected with SARS-CoV-2 Nucleocapsid protein expression vector containing a myc-His-tag® (Panel A) 
HEK-293T transfected with SARS-CoV-1 Nucleocapsid protein expression vector containing a myc-His-tag® (Panel B) HEK-293T transfected with MERS Nucleocapsid protein expression vector containing a myc-His-tag® (Panel C) HEK-293T transfected with control vector containing a myc-His-tag® (Panel D) with 271180 at 1/4000 dilution followed by ready to use Goat Anti-Rabbit IgG H&L (HRP polymer). Positive staining on (A) HEK-293T transfected with a SARS-CoV-2 Nucleocapsid protein expression vector is observed. No staining on (B) HEK-293T transfected with a SARS-CoV-1 Nucleocapsid protein expression vector, (C) HEK-293T transfected with MERS Nucleocapsid protein expression vector and (D) HEK-293T transfected with empty vector. The section was incubated with ab271180 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

Immunohistochemical analysis of paraffin-embedded Mouse lung labeling SARS-CoV-2 with ab271180 at 1/4000 dilution followed by ready to use Goat Anti-Rabbit IgG H&L (HRP polymer). Negative control: No staining on Mouse lung. The section was incubated with ab271180 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Goat Anti-Rabbit IgG H&L (HRP polymer).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

Immunohistochemical analysis of paraffin-embedded Rat lung labeling SARS-CoV-2 with ab271180 at 1/4000 dilution followed by ready to use Goat Anti-Rabbit IgG H&L (HRP polymer). Negative control: No staining on Rat lung. The section was incubated with ab271180 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Goat Anti-Rabbit IgG H&L (HRP polymer).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100-permeabilized 293T cells transfected with myc-tagged SARS-CoV-2 Nucleocapsid protein expression vector labelling SARS-CoV-2 with ab271180 at 1/1000 dilution, followed by ab150080 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 594) antibody at 1/1000 dilution (Red). ab202008 Anti-Myc tag mouse monoclonal antibody (Alexa Fluor® 488) was used to counterstain Myc tag at 1/200 dilution (Green). The nuclear counterstain was DAPI (Blue). Confocal image showing cytoplasmic staining in 293T cells transfected with myc-tagged SARS-CoV-2 Nucleocapsid protein expression vector.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100-permeabilized Vero cells labelling SARS-CoV-2 with ab271180 at 1/1000 dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) antibody at 1/500 dilution. Fluorescent image showing positve staining in Vero cells after infecting with SARS-CoV-2 virus for 48 h. Nuclear counter stain DAPI. 
Two indenpendent optical fields were captured for imaging at each given antibody dilution.

This piece of data was kindly provided by Dr. Hangping Yao, State Key Laboratory for Diagnosis and Treatment of Infectious Diseases in Zhejiang University, School of Medicine

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100-permeabilized 293T cells transfected with myc-tagged SARS-CoV-1 and MERS Nucleocapsid protein labelling SARS-CoV-2 with ab271180 at 1/1000 dilution, followed by ab150080 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 594) antibody at 1/1000 dilution. ab202008 Anti-Myc tag mouse monoclonal antibody (Alexa Fluor® 488) was used to counterstain Myc tag at 1/200 dilution (Green). The nuclear counterstain was DAPI (Blue). Confocal image showing no staining in 293T cells transfected with myc-tagged SARS-CoV-1 and MERS Nucleocapsid protein, which are negative controls for SARS-CoV-2.

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized HEK-293T (human embryonic kidney) transfected with myc-tagged SARS-CoV-2 Nucleocapsid protein expression construct  labelling SARS-CoV-2 with ab271180 at 1/500 dilution (Right) compared with a Rabbit monoclonal IgG (ab172730) (Left) isotype control. 

A Goat anti rabbit IgG (Alexa Fluor® 488, ab150077 at 1/2000 dilution was used as the secondary antibody.

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized HEK-293T (human embryonic kidney) transfected with myc-tagged SARS-CoV-1 Nucleocapsid protein expression construct (Left)/ HEK-293T (human embryonic kidney) transfected with myc-tagged MERS Nucleocapsid protein expression construct (Right). 

A Goat anti rabbit IgG (Alexa Fluor® 488, ab150077 at 1/2000 dilution was used as the secondary antibody.

SARS-CoV-2 was immunoprecipitated from 0.35 mg HEK-293T (human embryonic kidney) transfected with SARS-CoV-2 Nucleocapsid protein expression vector containing a myc-His-tag®, whole cell lysate 5 µg with ab271180 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab271180 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: HEK-293T (human embryonic kidney) transfected with SARS-CoV-2 Nucleocapsid protein expression vector containing a myc-His-tag® whole cell lysate 5 μg

Lane 2: ab271180 IP in HEK-293T transfected with SARS-CoV-2 Nucleocapsid protein expression vector containing a myc-His-tag® whole cell lysate.

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab271180 in HEK-293T transfected with SARS-CoV-2 Nucleocapsid protein expression vector containing a myc-His-tag® whole cell lysate

Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Exposure time: 3.25 seconds.

Plates were coated with Recombinant Human coronavirus SARS-CoV-2 nucleocapsid protein (His tag, ab273530) and Recombinant Human coronavirus SARS Nucleocapsid Protein (ab270829) at 500 ng/ml.

Primary antibody (ab271180) concentration range 0.045-100 ng /ml.

Pre-adsorbed secondary antibody, goat anti-rabbit IgG H&L (HRP, ab97080) used at 1/2000 dilution.